The long-term interest of this research program is to understand the cellular mechanisms of vascular remodeling in pulmonary hypertension. This proposal will focus on two major areas. First, it will address the controversial area of the activity and role of upregulated nitric oxide synthase in the cellular mechanisms of vascular remodeling. Second, it will investigate a novel protein, hypoxia-induced mitogenic factor (HIMF) we have discovered in the proliferating microvasculature of lungs from chronic hypoxia exposed animals. The first aim will address the hypothesis that, despite marked upregulation of NOS isoforms in chronic hypoxia-induced pulmonary hypertension, bio-available NO is reduced due to limitation of oxygen substrate by hypoxia, limitation of L-arginine substrate due to upregulation of L-arginase, and increased production of endogenous inhibitors of NOS. The second aim will investigate the effect of NO and L-arginase I expression on polyamine activation of p42/p44 protein kinases, p21 and resultant changes in cell cycle and proliferative response of pulmonary microvascular smooth muscle. It will investigate whether enhanced NO production through L-arginase inhibition or L-arginine administration, will increase NO production and restore its antiproliferative effect on pulmonary VSM in cultured cells, lung organ culture and whole animal. Aim 3 will address the hypothesis that hypoxia-induced mitogenic protein (HIMF) is a novel mitogenic factor in proliferating/remodeling pulmonary microvascular endothelial and vascular smooth muscles cells during the development of pulmonary hypertension. It will define the mitogenic/proliferative actions of this protein in intact chronic hypoxia lung models, lung organ culture, and isolated microvascular cells. It will determine the effect of blockade of HIMF on the development of pulmonary vascular remodeling and identify the receptors for HIMF. The fourth aim will address the hypothesis that L-arginase and HIMF are upregulated by hypoxia via C/EBP-beta and/or HIF-1-related mechanisms. It will utilize comprehensive promoter-reporter transfection studies, EMSA, foot printing, overexpression, and knockout studies to define the protein-DNA interactions critical to hypoxia-induced L-arginase and HIMF, and assess the role of posttranslational modification in the hypoxia response.